N-9 Guanine Nucleosides - American

Oct 25, 2010 - University of Delhi, Delhi-110 007, India, ‡University of. Copenhagen, Faculty of Life Sciences, Department of Natural. Sciences, DK-...
1 downloads 0 Views 887KB Size
Download PDF
pubs.acs.org/joc

Biocatalytic Separation of N-7/N-9 Guanine Nucleosides Sunil K. Singh,† Vivek K. Sharma,† Carl E. Olsen,‡ Jesper Wengel,§ Virinder S. Parmar,†,§ and Ashok K. Prasad*,† †

Bioorganic Laboratory, Department of Chemistry, University of Delhi, Delhi-110 007, India, ‡University of Copenhagen, Faculty of Life Sciences, Department of Natural Sciences, DK- 1871 Frederiksberg C, Denmark, and §Nucleic Acid Centre, Department of Physics and Chemistry, University of Southern Denmark, DK-5230 Odense M, Denmark [email protected] Received August 9, 2010

Vorbr€ uggen coupling of trimethylsilylated 2-N-isobutanoylguanine with peracetylated pentofuranose derivatives generally gives inseparable N-7/N-9 glycosyl mixtures. We have shown that the two isomers can be separated biocatalytically by Novozyme-435-mediated selective deacetylation of the 50 -O-acetyl group of peracetylated N-9 guanine nucleosides.

that are difficult to separate.3,4 The changes in experimental variables and the use of a selectively modified guanine moiety, such as 2-N-acetyl-6-O-diphenylcarbamoylguanine in nucleoside coupling reactions, affect the isomeric ratio but do not eliminate the formation of the N-7 isomer together with the desired N-9 isomer.5 In this paper, we report the synthesis of guanine nucleosides (mixture of 9- and 7-glycosyl derivatives) derived from D-ribose, D-arabinose, and L-arabinose sugars and for the first time their highly efficient separation mediated by Novozyme-435 lipase-catalyzed removal of one of the acetoxy functions of the peracetylated N-9 guanine nucleosides. The coupling of 2-N-isobutanoylguanine (2)6 with 1,2,3,5tetra-O-acetyl-D-ribofuranose (3),7,8 1,2,3,5-tetra-O-acetyl9 D-arabinofuranose (4), or 1,2,3,5-tetra-O-acetyl-L-arabino8-10 in the presence of TMSOTf as Lewis furanose (5) acid catalyst following a standard Vorbr€ uggen5,11 coupling protocol afforded mixtures of 2,3,5-tri-O-acetylated 9- and 7-β-D-ribofuranosylguanines 6 and 7, 9- and 7-R-D-arabinofuranosylguanines 8 and 9, and 9- and 7-R-L-arabinofuranosylguanines 10 and 11 in ratios of 87:13, 63:37, and 76:24, respectively, in 60-65% yields (Scheme 1 and Table 1). The ratio of regioisomers N-9 and N-7 in the above guanine nucleoside mixtures 6 and 7, 8 and 9, and 10 and 11 were calculated on the basis of the integration of the corresponding anomeric protons in the 1H NMR spectra (400 MHz) of the mixtures (Table 1). Our various attempts of separation of N-9 and N-7 guanine nucleosides from the mixtures 6 and 7, 8 and 9, and 10 and 11 by repeated column chromatography on silica gel were unsuccessful. Some lipases have been found to selectively acylate/deacylate primary hydroxyl over secondary hydroxyl group(s) of sugars12

(1) Elion, G. B.; Furman, P. A.; Fyfe, J. A.; Miranda, P. De.; Beauchamp, L.; Schaeffer, H. J. Proc. Natl. Acad. Sci. U.S.A. 1977, 74, 5716–5720. (2) (a) Herdewijn, P. Modified Nucleosides. In Biochemistry, Biotechnology and Medicine; Wiley-VCH: New York, 2008. (b) Chu, C. K. Antiviral Nucleosides: Chiral Synthesis and Chemotherapy; Elsevier: New York, 2003.

(3) Zhong, M.; Robins, M. J. Tetrahedron Lett. 2003, 44, 9327–9930. (4) (a) Schaffer, H. J.; Beauchamp, L.; Miranda, P. de.; Elion, G. B.; Bauer, D. J.; Collins, P. Nature 1978, 272, 583–585. (b) Robins, M. J.; Hatfield, P. W. Can. J. Chem. 1982, 60, 547–553. (c) Boryski, J.; Golankiewicz, B. Nucleosides Nucleotides 1987, 6, 385–386. (d) Matsumoto, H.; Kaneko, C.; Yamada, K.; Takeuchi, T.; Mori, T.; Mizuno, Y. Chem. Pharm. Bull. 1988, 36, 1153–1157. (e) Boryski, J. J. Chem. Soc., Perkin Trans. 2 1997, 649–652. (f ) Clair, A. S.; Xiang, G.; McLaughlin, L. W. Nucleosides Nucleotides 1998, 17, 925–937. (g) Boryski, J.; Golankiewicz, B. Nucleosides Nucleotides 1989, 8, 529–536. (h) Geen, G.; Grinter, T. J.; Kincey, P. M.; Jarvest, R. L. Tetrahedron 1990, 46, 6903–6914. (i) Izawa, K.; Shiragami, H. Pure Appl. Chem. 1998, 70, 313–318. ( j) Li, N. -S.; Piccirilli, J. A. Synthesis 2005, 17, 2685–2870. (k) Ferenc, G.; Kele, Z.; Kov acs, L. Rapid Commun. Mass Spectrom. 2005, 19, 236–240. (5) (a) Vorbr€ uggen, H.; Krolikiewicz, K.; Bennua, B. Chem. Ber. 1981, 114, 1234–1255. (b) Robins, M. J.; Zou, R.; Guo, Z.; Wnuk., S. F. J. Org. Chem. 1996, 61, 9207–9212. (c) Garner, P.; Ramakanth, S. J. Org. Chem. 1988, 53, 1294–1298. (d) Zou, R.; Robins, M. J. Can. J. Chem. 1987, 65, 1436–1437. (e) Cheung A. W.-H.; Sidduri, A.; Garofalo, L. M.; Goodnow, R. A., Jr. Tetrahedron Lett. 2000, 41, 3303–3307. (6) Milecki, J.; Foldesi, A.; Fischer, A.; Adamiak, R. W.; Chattopadhyaya, J. J. Labelled Compd. Radiopharm. 2001, 44, 763–783. (7) Wright, G. E.; Dudycz, L. W. J. Med. Chem. 1984, 27, 175–181. (8) Shi, C.-J.; Zhang, J.; Fu, J.; Tang, J. Lett. Org. Chem. 2006, 3, 932–935. (9) Gupta, P.; Maity, J.; Shakya, G.; Prasad, A. K.; Parmar, V. S.; Wengel, J. Org. Biomol. Chem. 2009, 7, 2389–2401. (10) Kam, B. L.; Barascut, J. L.; Imbach, J. L. Carbohydr. Res. 1979, 69, 135–142. (11) Vorbr€ uggen, H.; H€ ofle, G. Chem. Ber. 1981, 114, 1256–1268. (12) (a) Prasad, A. K.; Kalra, N.; Yadav, Y.; Kumar, R.; Sharma, S. K.; Patkar, S.; Lange, L.; Wengel, J.; Parmar, V. S. Chem. Commun. 2007, 2616– 2617. (b) Maity, J.; Shakya, G.; Singh, S. K.; Ravikumar, V. T.; Parmar, V. S.; Prasad, A. K. J. Org. Chem. 2008, 73, 5629–5632. (c) Sharma, R. K.; Aggarwal, N.; Arya, A.; Olsen, C. E.; Parmar, V. S.; Prasad, A. K. Indian J. Chem. 2009, 48B, 1727–1731.

7932

Published on Web 10/25/2010

The discovery of 9-[(2-hydroxyethoxy)methyl]guanine (acyclovir) against herpes simplex type 1 and type 2 viruses and its low mammalian toxicity triggered the synthesis of a series of guanine nucleosides, e.g., penciclovir, famciclovir, valaciclovir, valganciclovir, abacavir, etc., for the treatment of various viral diseases.1,2 The most problematic chemistry and difficulties in manipulation of all five common bases found in DNA and RNA occur with the polyfunctional guanine (pKa1 -1.7, pKa2 -9.2) nucleosides and nucleotides. The coupling of guanine with peracetylated sugar derivatives generally produces N-7/N-9 isomeric mixtures of nucleosides

J. Org. Chem. 2010, 75, 7932–7935

DOI: 10.1021/jo101565e r 2010 American Chemical Society

JOC Note

Singh et al. SCHEME 1.

Procedure for the Synthesis of Guanine Nucleosides

FIGURE 1. Selective deacetylation studies on nucleosides 6 and 7, TABLE 1.

Glycosylation of 2-N-Isobutanoylguanine with Peracetylated Sugar Derivatives 3-5 reaction ratioa of N-9/ N-7 yields reactants time (h) products nucleosides (%) 2þ3 2þ4 2þ5

4 6 6

6 and 7 8 and 9 10 and 11

87:13 63:37 76:24

60 62 65

8 and 9, and 10 and 11 in THF at 40-42 °C catalyzed by Novozyme435 and other lipases.

SCHEME 2. Novozyme-435-Catalyzed Deacetylation Reaction: Separation of N-7 and N-9 Guanine Nucleosides

a Ratios of N-9 and N-7 nucleoside regioisomers were calculated from the 1H NMR spectral (400 MHz) data of the mixtures.

and nucleosides.13 In the recent past, lipases have been used for the separation of the nucleosides from their anomeric mixtures14 and for the resolution of β-D/L-20 - deoxynucleosides.15 Stimulated by these results, we decided to explore an enzymatic route for the separation of N-9 and N-7 guanine nucleosides. Five lipases, viz. Candida antarctica lipase-B immobilized on polyacrylate, Lewatit (Novozyme-435 or CAL-B), Theremomyces lanuginosus lipase immobilized on silica (Lipozyme TL IM), Amano PS lipase, Candida rugosa lipase (CRL), and porcine pancreatic lipase (PPL) were screened for the selective deacetylation of one regioisomer over the other in N-9/N-7 guanine nucleoside mixtures 6 and 7, 8 and 9, and 10 and 11 in five sets of organic solvents, i.e., tetrahydrofuran (THF), acetonitrile (CH3CN), toluene, diisopropyl ether (DIPE), and acetone using n-butanol as acetyl acceptor at 40-42 °C and at 150 rpm in an incubator shaker (Figure 1). It was observed that lipase Novozyme-435 in THF at 40-42 °C selectively and most efficiently deacetylates the acetoxy group at the C-40 -hydroxymethyl moiety of N-9glycosylguanine over the other acetoxy groups of N-9 and N-7 isomers of nucleosides (Scheme 2). The Novozyme-435catalyzed deacetylation reaction carried out in other solvents, i.e., acetonitrile, toluene, DIPE, and acetone, did not yield any product. Although PPL in THF initially showed selectivity for the deacetylation of the C-40 -acetoxymethyl group of N-9-glycosylguanine, the reaction was too slow to be of any practical use, and deacetylation of other acetoxy groups of the N-7 and N-9 regioisomers were observed leading to the formation of a mixture of compounds. When the Novozyme-435 catalyzed reaction was conducted at a higher temperature than 40-42 °C, the enhancement in the rate was observed, but it resulted in the formation of multiple (13) (a) Prasad, A. K.; Trikha, S.; Parmar, V. S. Bioorg. Chem. 1999, 27, 135–154. (b) Ciuffreda, P.; Casati, P.; Santaniello, E. Bioorg. Med. Chem. Lett. 1999, 9, 1577–1582. (c) Ferrero, M.; Gotor, V. Chem. Rev. 2000, 100, 4319–4347. (14) (a) Damkjaer, D. L.; Petersen, M.; Wengel, J. Nucleosides Nucleotides 1994, 13, 1801–1807. (b) Garcia, J.; Diaz-Rodriguez, A.; Fernandez, S.; Sanghvi, Y.; Ferrero, M.; Gotor, V. J. Org. Chem. 2006, 71, 9765–9771. (15) Garcia, J.; Fernandez, S.; Ferrero, M.; Sanghvi, Y.; Gotor, V. Org. Lett. 2004, 6, 3759–3762.

products. Lipozyme TL IM, Amano PS, and CRL did not show any reaction on the peracetylated N-9-, N-7-glycosylguanine nucleoside substrate mixtures. In a typical reaction, a solution of N-9/N-7 nucleoside mixture 6 and 7 (200 mg) in tetrahydrofuran (20 mL) containing a small amount of n-butanol was incubated with Novozyme435 (substrate-enzyme ratio 1:0.5 w/w) in an incubator shaker at 40-42 °C. The reaction mixture was shaken for 4-6 h until a prominent spot of the product appeared on analytical TLC at much lower Rf value than the starting compounds.16 The reaction was quenched by filtering off the enzyme, and the solvent was removed under reduced pressure. The crude product thus obtained was purified by silica gel column chromatography using methanol in chloroform as gradient solvent system to afford the pure N-9 glycosylguanine 12 having a C-50 hydroxyl group in 72% yield (Scheme 2). Similarly, lipase-catalyzed deacetylation reactions on nucleoside mixtures 8 and 9 and 10 and 11 led to the formation of the selectively deacetylated compounds 13 and 14 in 50 and 61% yields, respectively, along with the unreacted peracetylated pure N-7 glycosylated guanines 9 and 11 in 30 and 20% yields, respectively.17 These reactions did not yield any product when carried out in the absence of lipase. All the enzymatically deacetylated N-9 glycosylguanine nucleosides 12-14 (16) Completion of the reaction was monitored by TLC in methanol-chloroform as solvent system. Partial separation in N-9- and N-7-glycosylated nucleosides was observed after five to six runs of the TLC plate in the solvent. (17) Yields of N-9 glycosylated guanines 12-14 and N-7 glycosylated guanines 9 and 11 were calculated on the basis of the consideration of N-9 and N-7 regioisomers as 100% in the mixtures.

J. Org. Chem. Vol. 75, No. 22, 2010

7933

JOC Note SCHEME 3.

Singh et al. Deacetylation of Guanine Nucleosides 12-14

and unreacted, recovered peracetylated N-7 glycosylguanine nucleosides 9 and 11 were unambiguously identified on the basis of their spectral (IR, 1H and 13C NMR) and HRMS data. The 50 -OH group of selectively deacetylated N-9-glycosylated guanine nucleosides 12-14 was acetylated using acetic anhydride and DMAP as a catalyst in THF resulting in the formation of 20 ,30 ,50 -tri-O-acetylated guanine nucleosides 6, 8, and 10 in 82, 84, and 85% yields, respectively. The NMR spectral data of pure tri-O-acetylated N-9 glycosylguanines 6,18 8, and 10 and pure N-7 glycosylguanines 9 and 11 were compared with the NMR spectral data of N-9/N-7 glycosylated guanine mixtures 6 and 7, 8 and 9, and 10 and 11 to further confirm the identity of the individual compounds. To confirm the structures of Novozyme-435 deacetylated nucleosides, hydrolysis of compounds 12-14 was performed in methanolic ammonia to yield guanosine (15),5 R-D-arabinofuranosylguanine 16,9 and R-L-arabinofuranosylguanine 17 in 90, 92, and 95% yields, respectively (Scheme 3). The hydrolyzed products 15-17 were unambiguously identified on the basis of their spectral (IR, 1H and 13C NMR spectra) and HRMS data analysis. The physical and spectral data of known compounds 15 and 16 were found to be identical to the reported data; the spectral data of compound 17 has not been previously reported. In summary, it has been established that the lipase Novozyme-435 can selectively hydrolyze the acetoxy function derived from the primary hydroxyl group at the C-50 -position of tri-O-acetylated N-9 glycosylated guanines over the acetoxy moieties derived from the two secondary hydroxyl groups present in the same molecule and also over three acetoxy functions of N-7 glycosylguanines. This enzymatic methodology enables efficient separation of mixtures of N-9 and N-7 glycosylated guanines, which are otherwise almost impossible to separate by column chromatography or other techniques. Experimental Section General Procedure of Lipase-Catalyzed Selective Deacetylation of Mixtures of N-9/N-7 Nucleosides 6 and 7, 8 and 9, and 10 (18) Rigoli, J. W.; Østergaard, M. E.; Canady, K. M.; Guenther, D. C.; Hrdlicka, P. J. Tetrahedron Lett. 2009, 50, 1751–1753.

7934

J. Org. Chem. Vol. 75, No. 22, 2010

and 11. The solution of N-9/N-7 guanine nucleosides 6 and 7, 8 and 9, or 10 and 11 (200 mg, 0.42 mmol) in tetrahydrofuran (20 mL) containing a small amount of n-butanol as acetyl acceptor was incubated with C. antarctica lipase B (Novozyme-435, 100 mg) in an incubator shaker at 40-42 °C. After 4-6 h of incubation, a prominent spot of the product appeared at much lower Rf value than the starting compounds.16 The reaction was stopped by filtering off the enzyme, and the solvent was removed under reduced pressure. The crude product thus obtained was purified through a silica gel column using a gradient solvent system of methanol in chloroform (1:99 to 1.5:98.5) to afford pure 50 hydroxylated N-9 glycosylguanine nucleosides 12, 13, and 14 in 72%, 50%, and 61% yields, respectively. The recovered unreacted N-7 guanine nucleosides 9 and 11 were isolated in pure form in 30 and 20% yields, respectively, whereas the recovered N-7 nucleoside 7 was contaminated with N-9 guanine nucleoside. 9-(20 ,30 -Di-O-acetyl-β-D-ribofuranosyl)-N2-isobutanoylguanine (12). Compound 12 was obtained as a white sticky solid (0.13 g, 72% yield): Rf = 0.5 (10% methanol in chloroform); [R]25D = þ2.2 (c 0.07, methanol); IR (KBr) νmax 3220, 2936, 1751, 1684, 1609, 1560, 1405, 1375, 1250, 1157, 1105, 949, 785 cm-1; 1H NMR (CD3OD, 400 MHz) δ 1.23 (6H, d, J = 6.8 Hz), 2.03, 2.12 (6H, 2s), 2.70-2.73 (1H, m), 3.78-3.88 (2H, m), 4.26-4.28 (1H, m), 5.56-5.58 (1H, m), 5.82 (1H, t, J = 5.6 Hz), 6.18 (1H, d, J = 6.0 Hz), 8.29 (1H, s); 13C NMR (CD3OD, 100.6 MHz): δ 19.36, 20.11, 20.50, 37.03, 62.35, 72.78, 75.38, 87.11, 85.34, 121.44, 139.74, 150.07, 150.63, 157.49, 171.11, 171.64, 181.81; HR-ESI-TOF-MS m/z 460.1432 ([M þ Na]þ), calcd for [C18H23N5O8 þ Na]þ 460.1439. 9-(20 ,30 -Di-O-acetyl-r-D-arabinofuranosyl)-N2-isobutanoylguanine (13). Compound 13 was obtained as a white solid (90 mg, 50% yield): Rf = 0.5 (10% methanol in chloroform); mp 180-182 °C; [R]25D = þ3.9 (c 0.1, methanol); IR (KBr) νmax 3196, 2975, 2935, 1737, 1718, 1690, 1615, 1567, 1370, 1247, 1137, 1043, 908, 800 cm-1; 1H NMR (CD3OD, 400 MHz): δ 1.22 (6H, d, J = 6.8 Hz), 2.05, 2.09 (6H, 2s), 2.71-2.74 (1H, m), 3.73-3.83 (2H, m), 4.56-4.59 (1H, m), 5.43-5.45 (1H, m), 5.85 (1H, t, J = 4.0 Hz), 6.22 (1H, d, J = 3.6 Hz) and 8.13 (1H, s); 13C NMR (CD3OD, 100.6 MHz) δ 19.36, 20.02, 20.05, 36.99, 62.41, 76.93, 81.08, 86.16, 89.04, 121.65, 139.85, 150.06, 150.68, 157.49, 170.54, 171.49, 181.87; HR-ESITOF-MS m/z 460.1426 ([M þ Na]þ), calcd for [C18H23N5O8 þ Na]þ 460.1439. 9-(20 ,30 -Di-O-acetyl-r-L-arabinofuranosyl)-N2-isobutanoylguanine (14). Compound 14 was obtained as a white solid (0.11 g, 61% yield): Rf = 0.5 (10% methanol in chloroform); mp 176-178 °C; [R]25D = -4.2 (c 0.1, methanol); IR (KBr) νmax 3193, 2976, 2950, 1737, 1719, 1691, 1612, 1566, 1407, 1370, 1247, 1137, 1071, 909, 801 cm-1; 1H NMR (CD3OD, 400 MHz) δ 1.22 (6H, d, J = 6.8 Hz), 2.09, 2.05 (6H, 2s), 2.71-2.74 (1H, m), 3.76-3.80 (2H, m), 4.57-4.59 (1H, m), 5.43-4.45 (1H, m), 5.85 (1H, t, J = 4.0 Hz), 6.22 (1H, d, J = 4.0 Hz), 8.13 (1H, s); 13C NMR (CD3OD, 100.6 MHz) δ 19.36, 20.02, 20.05, 36.99, 62.41, 76.93, 81.08, 86.16, 89.04, 121.65, 139.85, 150.06, 150.68, 157.49, 170.54, 171.49, 181.87; HR-ESI-TOF-MS m/z 460.1426 ([M þ Na]þ), calcd for [C18H23N5O8 þ Na]þ 460.1439. 7-(20 ,30 ,50 -Tri-O-acetyl-r-D-arabinofuranosyl)-N2-isobutanoylguanine (9). Compound 9 was obtained as a white sticky solid (59 mg, 30% yield): Rf = 0.5 (7% methanol in chloroform); [R]25D = þ1.7 (c 0.1, methanol); IR (KBr) νmax 3188, 2976, 1751, 1686, 1610, 1375, 1226, 1054, 781 cm-1; 1H NMR (CD3OD, 400 MHz) δ 1.21 (6H, d, J = 6.8 Hz), 1.99-2.10 (9H, 3s), 2.70-2.74 (1H, m), 4.27-4.40 (2H, m), 4.85-4.86 (1H, m), 5.37-5.39 (1H, m), 5.88 (1H, t, J = 4.0 Hz), 6.41 (1H, d, J = 4.0 Hz), 8.35 (1H, s); 13C NMR (CD3OD, 100.6 MHz) δ 19.36, 20.13, 20.70, 20.92, 36.96, 64.39, 76.98, 81.59, 83.35, 91.38, 112.03, 144.30, 149.61, 154.81, 160.03, 171.51, 172.39,

Singh et al. 181.93; HR-ESI-TOF-MS m/z 502.1535 ([M þ Na]þ), calcd for [C20H25N5O9 þ Na]þ 502.1544. 7-(20 ,30 ,50 -Tri-O-acetyl-r-L-arabinofuranosyl)-N2-isobutanoylguanine (11). Compound 11 was obtained as a white sticky solid (40 mg, 20% yield): Rf = 0.5 (7% methanol in chloroform); [R]25D = -1.5 (c 0.1, methanol); IR (KBr) νmax 3434, 2927, 1751, 1685, 1611, 1551, 1372, 1224, 1055, 781 cm-1; 1H NMR (CDCl3, 300 MHz) δ 1.24 (6H, d, J = 6.8 Hz), 2.03-2.15 (9H, 3s), 2.83-2.85 (1H, m), 4.29-4.38 (2H, m), 4.79 (1H, brs), 5.31 (1H, t, J = 3.6 Hz), 5.83 (1H, t, J = 3.2 Hz), 6.43 (1H, d, J = 3.0 Hz), 8.01 (1H, s), 9.86 (1H, brs), 12.36 (1H, brs); 13C NMR (CDCl3, 75.5 MHz) δ 19.05, 20.68, 20.79, 36.15, 62.95, 75.96, 80.36, 82.55, 90.26, 111.31, 141.58, 148.14, 152.99, 157.84, 169.36, 169.74, 170.61, 179.65; HR-ESI-TOF-MS m/z 502.1537 ([M þ Na]þ), calcd for [C20H25N5O9 þ Na]þ 502.1544. General Procedure of Acetylation of Enzymatically Selectively Deacetylated Guanine Nucleosides 12-14. To a stirred solution of nucleosides 12-14 (0.1 g, 0.21 mmol) in THF (8 mL) was added acetic anhydride (0.25 mmol), followed by the addition of catalytic amount of DMAP, and the reaction mixture was stirred for 4-5 h at 28 °C. On completion (examination on analytical TLC), crushed ice was added, and the reaction mixture was extracted with ethyl acetate (2  30 mL). The combined organic layer was washed with saturated aqueous NaHCO3 solution (2  30 mL) and dried over anhydrous Na2SO4, and the solvent was removed under reduced pressure. The residue thus obtained was purified by column chromatography on silica gel using methanol/chloroform (1:99) as eluent to afford the pure triacetylated guanine nucleosides 6,17 8, and 10 in 82, 84, and 85% yields, respectively. 9-(20 ,30 ,50 -Tri-O-acetyl-β-D-ribofuranosyl)-N2-isobutanoylguanine (6)18. Compound 6 was obtained as a white sticky solid (90 mg, 82% yield): Rf = 0.5 (7% methanol in chloroform); [R]25D = þ5.2 (c 0.1, methanol); HR-ESI-TOF-MS m/z 502.1533 ([M þ Na]þ), calcd for [C20H25N5O9 þ Na]þ 502.1544. 9-(20 ,30 ,50 -Tri-O-acetyl-r-D-arabinofuranosyl)-N2-isobutanoylguanine (8). Compound 8 was obtained as a white sticky solid (92 mg, 84% yield): Rf = 0.5 (7% methanol in chloroform); [R]25D = þ4.2 (c 0.1, methanol); IR (KBr) νmax 3167, 2976, 2936, 1749, 1683, 1609, 1560, 1403, 1373, 1224, 1101, 1053, 907, 785 cm-1; 1H NMR (CD3OD, 400 MHz) δ 1.22 (6H, d, J = 6.8 Hz), 2.05, 2.06, 2.10 (9H, 3s), 2.73 (1H, m), 4.32-4.36 (2H, m), 4.79-4.86 (1H, m), 5.36-5.38 (1H, m), 5.84 (1H, t, J = 3.6 Hz), 6.23 (1H, d, J = 3.2 Hz), 8.11 (1H, s); 13C NMR (CD3OD, 100.6 MHz) δ 19.36, 20.54, 20.68, 37.01, 64.07, 77.17, 80.79, 83.56, 89.23, 121.67, 139.60, 150.07, 150.37, 157.48, 171.29, 171.38, 172.39, 181.86; HR-ESI-TOF-MS m/z 502.1526 ([M þ Na]þ), calcd for [C20H25N5O9 þ Na]þ 502.1544. 9-(20 ,30 ,50 -Tri-O-acetyl-r-L-arabinofuranosyl)-N2-isobutanoylguanine (10). Compound 10 was obtained as a white sticky solid (93 mg, 85% yield): Rf = 0.5 (7% methanol in chloroform); [R]25 D = -4.6 (c 0.1, methanol); IR (thin film) νmax 3153, 2928, 1747, 1683, 1609, 1563, 1402, 1373, 1225, 1158, 1053, 908, 757 cm-1; 1H NMR (CD3OD, 400 MHz) δ 1.22 (6H, d, J = 6.8 Hz), 2.05, 2.06,

JOC Note 2.10 (9H, 3s), 2.73 (1H, m), 4.32-4.36 (2H, m), 4.79-4.86 (1H, m), 5.36-5.38 (1H, m), 5.83-5.85 (1H, m), 6.23 (1H, d, J = 3.2 Hz), 7.90 (1H, s); 13C NMR (CD3OD, 100.6 MHz) δ 19.36, 20.54, 20.68, 37.01, 64.07, 77.17, 80.79, 83.56, 89.23, 121.67, 139.60, 150.07, 150.37, 157.48, 171.29, 171.38, 172.39, 181.86; HR-ESI-TOF-MS m/z 502.1538 ([M þ Na]þ), calcd for [C20H25N5O9 þ Na]þ 502.1544. General Procedure for the Hydrolysis of Nucleosides 12-14. A solution of selectively deacetylated nucleosides 12-14 (0.1 g, 0.22 mmol) in 20% methanolic ammonia solution (15 mL) was stirred at 25-28 °C for 10 h. On completion of the reaction (examination on analytical TLC), the excess solvent was removed under reduced pressure and the residue thus obtained was washed with hot ethyl acetate several times to afford guanosine (15),5 9-R-D-arabinofuranosyl guanine (16),9 and 9-R-L-arabinofuranosyl guanine (17) as white solids in 90, 92, and 95% yields, respectively. Guanosine (15)5. Compound 15 was obtained as a white solid (57.6 mg, 90% yield): mp 238-240 °C dec (lit.5 mp 240 °C dec); [R]25D = -40.7 (c 0.1, CH3SOCH3); HR-ESI-TOF-MS m/z 284.0988 ([M þ H]þ), calcd for [C10H13N5O5 þ H]þ 284.0989. 9-r-D-Arabinofuranosylguanine (16)9. Compound 16 was obtained as a white solid (58.9 mg, 92% yield): Rf = 0.3 (25% methanol in chloroform); mp 210 °C dec; [R]32D = þ21.8 (c 0.1, MeOH); HR-ESI-TOF-MS m/z 306.0804 ([M þ Na]þ), calcd for [C10H13N5O5 þ Na]þ 306.0809. 9-r-L-Arabinofuranosylguanine (17). Compound 17 was obtained as a white solid (60.8 mg, 95% yield); Rf = 0.3 (25% methanol in chloroform); mp 238 °C dec; [R]32D = -25.6 (c 0.1, MeOH); IR (KBr) νmax 3368, 3200, 2933, 1729, 1693, 1609, 1534, 1398, 1052, 781 cm-1; 1H NMR (CD3OD, 400 MHz) δ 3.44-3.59 (2H, m), 3.93-3.96 (1H, m), 4.06-4.10 (1H, m), 4.46-4.48 (1H, m), 5.67 (1H, d, J = 4.8 Hz), 7.91 (1H, s); 13C NMR (CD3OD, 100.6 MHz): δ 60.90, 75.03, 79.38, 84.84, 87.31, 116.53, 135.96, 151.09, 153.32, 156.26; ESI-TOF-MS m/z 306.0805 ([M þ Na]þ), calcd for [C10H13N5O5 þ Na]þ 306.0809.

Acknowledgment. We acknowledge the financial support from the University of Delhi (DU-DST Purse Grant to A.K.P. and V.S.P.) and the DBT, New Delhi (under the Indo-Danish collaboration in Biotechnology to A.K.P., V.S.P., and J.W.). S.K.S. and V.K.S. thank the Council of Scientific and Industrial Research (CSIR, New Delhi) for the award of research fellowships. J.W. thanks the Danish National Research Foundation for financial support. Supporting Information Available: General experimental methods, procedure for the preparation of compounds 6 and 7, 8 and 9, and 10 and 11, and 1H- and 13C NMR spectra of mixtures of 6 and 7, 8 and 9, and 10 and 11, 6, and 8-17. This material is available free of charge via the Internet at http://pubs.acs.org.

J. Org. Chem. Vol. 75, No. 22, 2010

7935